The penultimate step of the coagulation cascade is the Factor Xa-complex-catalyzed conversion of prothrombin to thrombin. Prothrombin is a single-chain, vitamin K-dependent glycoprotein that is synthesized in the liver. Prothrombin contains a pro peptide, a gla domain, two kringle regions, an A chain and a serine protease domain. The A chain is disulfide-linked to the serine protease domain. Conversion to thrombin requires that prothrombin be cleaved in two places by Factor Xa-complex. One factor Xa cleavage liberates a protein fragment comprising the gla domain and two kringle regions. The other factor Xa cleavage, which is responsible for producing a catalytically active molecule, cleaves the A chain from the serine protease domain to form a disulfide-linked two chain protein. Cleavages at both factor Xa cleavage sites result in the formation of active thrombin which is composed of the 49 amino acid A chain disulfide-bonded to the serine protease domain. Thrombin hydrolyses specific arginyl-glycine bonds in fibrinogen to produce fibrin monomers, which self-assemble into a fibrin clot, and in Factor XIII to produce Factor XIIIa, which in turn cross-links the fibrin monomers to strengthen and stabilize the fibrin clot. The role of thrombin in the coagulation cascade has been reviewed, for example by Jackson and Nemerson (Ann. Rev. Biochem. 49: 765-811 (1980)).
Thrombin is used clinically to control bleeding during surgery, for burns and in certain trauma situations (Nakamura et al. The Amer. Surgeon 57:226-230 (1991); Thompson et al. Ophthalmologly 93:279-282 (1986); Harris et al., J. Bone Joint Surg. Am! 60:454-456 (1978); Craig and Asher, Spine 2:313-317 (1977); Prasad et al. Burns 17:70-71 (1991)). Bovine thrombin is also a component of some commercial tissue glues.
Commercial thrombin therapeutics are purified from pooled human and animal blood products and as such run the risk of contamination with viruses such as the HIV and hepatitis viruses. In comparing three commercial thrombin preparations, Suzuki and Sakuragawa (Thromb. Res. 53:271-278, 1989) found that the preparations contained contaminating proteins, and the human preparation contained immunoglobulin G, hepatitis B surface antigen antibodies and human immunodeficiency antibodies. Xenogeneic immunization with bovine thrombin has been reported in patients who have developed self-reactive antibodies to both human thrombin and human factor V (factor V is a contaminant in the bovine thrombin preparation) (Stricker et al., Blood 72:1375-1380 (1988); Flaherty and Weiner, Blood 73: 1388 (1989); Flaherty et al., Ann Int. Med. 111:631-634 (1989); Zehnder and Leung, Blood 76:2011-2016 (1990); Lawson et al., Blood 76:2249-2257 (1990); Stricker et al., Blood 72:1375-1380 (1988); Berguer et al., J. Trauma 31:408-411 (1991)). In addition, concerns have recently been raised regarding the possible contamination of bovine products with pathogens such as the bovine spongeform encephalitis agent, which is not detectable or inactivatable by conventional means. Therapeutic human blood products are also subject to contamination by viral particles such as the hepatitis virus and the human immunodeficiency virus.
Although prothrombin has been prepared through recombinant means, approximately 14% of the protein was abnormally carboxylated.
There is therefore a need in the art for methods for producing thrombin that is essentially free of contaminating proteins. The present invention fulfills this need and provides other related advantages.